The Protective Effect Of Selenium On Subchronic Toxicity Induced By DEHP In Rats

Phthalates（PAEs）are esterified derivatives of phthalates that are widely added in the manufacture of plastics.Among the plasticizers,di（2-ethylhexyl）phthalate（DEHP）is the most commonly used.DEHP is used to increase the flexibility of polyvinyl chloride（PVC）in consumer products such as construction materials,decoration materials,medical products,food containers,toys and clothing.The annual global DEHP production is expected to be between 2205 and 881.8 billion pounds^[1].Due to the weak chemical binding of DEHP to plastic products,the dissociated DEHP is widely present in the environment.Due to the different amounts and types of products containing DEHP,exposure can occur through oral ingestion,inhalation,skin contact and intravenous exposure through medical procedures.DEHP and its metabolites have been found in many human tissues and fluids,including urine,serum,cord blood,breast milk,amniotic fluid,and follicular fluid.The presence of DEHP in follicular fluid indicates that it can reach this nerve,and its presence in amniotic fluid and cord blood suggests that it can reach the fetus.Human exposure to DEHP is worrying because it is a known endocrine disrupting chemical and reproductive toxicant.The study found that neonatal cryptorchidism,hypospadias,and testicular cancer,decreased sperm quality in adult males,decreased male anogenital distance（AGD）and testosterone（T）levels were associated with exposure to DEHP.In addition,prenatal exposure to DEHP and endometriosis,precocious puberty,loss of early pregnancy,and abnormal sexual differentiation were associated with low birth weight.Therefore,DEHP exposure to human health risks has attracted considerable attention.The mechanism of DEHP testotoxicity is currently unclear,mainly including:PPAR activation pathway,estrogen receptor-mediated mechanism,oncogene activation,and affecting spermatogenesis by changing the activity of enzymes responsible for sperm maturation,etc.There is an increasing number of possibilities for inducing oxidative stress.Oxidative stress is the imbalance between the formation of reactive oxygen species（ROS）and antioxidant defense mechanisms.ROS plays an important role in the regulation of several important physiological functions,but it also explains changes that may be harmful to cells,such as ROS leading to cell damage,apoptosis and cell death.Oxidative stress is also associated with germ cell apoptosis and male infertility.The essential trace element selenium（Se）is a key component of cellular antioxidant defenses and is involved in regulating the redox balance of various forms of cellular selenoproteins in cells.Se is involved in basic biological processes from the cell’s antioxidant defense to the protection and repair of DNA and apoptosis.Se is also essential for the production of normal sperm and therefore plays a key role in reproduction.Considering the importance of Se in testis structure and function,the frequency of selenium deficiency and the high possibility of DEHP exposure,DEHP was used to test 8-week-old male Sprague-Dawley rats for 8 weeks and yeast Se was used.Intervention was made to focus on the observation of its reproductive toxicity,to study the reproductive toxicity of DEHP subchronic exposure to rats,and to investigate the possible protective effect of selenium on phthalate toxicity.Methods70 healthy male Sprague-Dawley rats were fed for 3 days and randomly divided into 7 groups according to body weight,10 in each group.In the control group and the three DEHP exposure groups,the doses were 300,600,and 900 mg/kg b.wt and the corresponding DEHP exposure doses of the three selenium intervention groups,ie,in the three DEHP exposure groups.Separately add 1mg/kg yeast selenium（produced by Jiangsu Pharmaceutical Co.,Ltd.）.DEHP and yeast selenium are evenly mixed in the basal feed according to the corresponding proportions to make a pellet feed.The feed is the average daily animal weight.10%of the food intake was given to the rats in groups and fed continuously for 8 weeks.During exposure,rats are free to drink water and feed.Effects of sub-chronic exposure to DEHP and selenium on general indicators of SD ratsObservations of the general state of the rats during exposure（eg,appearance,fur,drinking,feeding,urine,and death,etc.）;weekly The body weight,food intake,and food return were recorded and the total food utilization was calculated.The liver,kidney,spleen,testis,and epididymis were weighed and the organ coefficients were calculated,and statistical analysis was performed.Effect of sub-chronic exposure of DEHP and selenium on blood biochemistry and blood cell parameters of SD rats At the end of the last exposure period,the rats were fasted for 16 hours and then weighed to record their last fasting body weight.Blood was taken from the orbital venous plexus and 3.5 ml of biochemical serum was pipetted to collect 3.5 ml of rat whole blood.Total protein（TP）,albumin（ALB）,globulin（GLB）,and aspartate aminotransferase were detected using an AU640 automatic biochemical analyzer.AST),alanine aminotransferase（ALT）,serum urea nitrogen（BUN）,serum creatinine（Cre）,alkaline phosphatase（ALP）,serum triglyceride（TG）,serum glucose（GLU）,total cholesterol（CHOL）Eleven biochemical indicators.2 At the end of the last exposure period,the rats were fasted for 16 hours and the weight of the last fasting body weight was recorded.Blood was taken from the orbital venous plexus and 1.0ml of rat whole blood was taken with a 1.5ml EP tube to which a drop of EDTA anticoagulant was added.Automated blood analysis was performed using an ADVIA 2120i automatic five-class hematology analyzer.Determination of the total number of white blood cells（WBC）,neutrophils（NEUT）,eosinophils（EOS）,basophils（BASO）,monocytes（MONO）,lymphocytes（LYM）,total number of red blood cells（RBC）Hb,HGB,MCV,HCT,MCH,RDW,MCHC,and CMCH,Hemoglobin content distribution width（HDW）,total platelet count（PLT）,mean platelet volume（MPV）,platelet distribution width（PDW）,platelet volume（PCT）and unstained large cell number（LUC）and their corresponding percentages,etc.index.SPSS statistical software analysis results.Effects of sub-chronic exposure to DEHP and selenium on sperm motility and serum T in SD ratsEffects of DEHP and selenium on rat sperm motility:Rat epididymides were used to prepare sperm suspensions and computer-assisted sperm analysis was used.CASA measures the movement parameters of the sperm,including:VCL,VSL,VAP,ALH,LIN,STR,BCF,ELON,etc.;2DEHP and selenium influence on serum T:At the end of the last exposure period,the rats were fasted for 16 hours.Weigh and record the last fasting weight.Blood was taken from the orbital venous plexus and 3.5 ml of biochemical serum was used to remove 3.5 ml of rat whole blood.The serum was separated by centrifugation at 3000 r/min for 10 minutes.The remaining serum was aspirated to a new EP tube,and testosterone levels were determined by radioimmunoassay.Effect of sub-chronic exposure of DEHP and selenium on testicular enzyme and oxidation index of SD rats The right testis tissue was prepared into homogenate of testis using ultrasonic homogenizer,and the supernatant was frozen at-20℃ for centrifugation.GSH-Px,SOD,MDA,ACP,ALP,Lactate dehydrogenase（LDH）,succinate dehydrogenase（SDH）,and protein concentration determination.ResultsEffect of sub-chronic exposure to DEHP and selenium on general indices of SD rats During the exposure period,no significant abnormalities were observed in the control group,300 and 600 mg/kg dose groups compared with the control group;and 900 The rats in the mg/kg group experienced localized hair loss（limbs and abdomen）;the body weight of each treated group decreased slightly with increasing dose,and the 900 mg/kg group had significant differences compared to the control group.Difference（P<0.05）.The body weight of each selenium intervention group was increased compared with the same dose of DEHP treated group,and the body weight of Se+ 300 mg/kg intervention group was significantly increased compared with the same dose of DEHP treated group（P<0.05）.0.05);When the exposure dose was increased to 900mg/kg,the organ coefficient of liver,kidney,spleen,testis,and epididymis of rats showed significant differences compared with the control group,and the difference was statistically significant（P<0.05 or P<0.01）.Effect of sub-chronic exposure to DEHP and selenium on blood biochemistry and blood cell parameters in SD rats Effect of blood biochemical indicators:Biochemical indicators related to liver and kidney were dosed with 300and 600 mg/kg DEHP doses There were abnormalities;among the biochemical indicators related to lipid and carbohydrate metabolism,rat TG and CHOL gradually decreased with increasing dose,and there was a statistically significant difference between the 900 mg/kg DEHP group and the control group.P<0.05).GLU in rats increased with the increase of the dose,and the difference between Se+600 and Se+900 mg/kg intervention groups was statistically significant（P<0.01）.2Implications of blood cells in blood:600 and 900mg/kg DEHP exposure groups,rat leukocytes（WBC,MONO,LUC）,red blood cells（RBC,HGB,HCT,MCH,HDW）and platelet PLT-related indicators abnormalities,and selenium intervention Changes in white blood cells are obvious,and little change is made in red blood cells and platelets.Effects of sub-chronic exposure to DEHP and selenium on sperm motility and serum T in SD rats With the increase of DEHP dose,VAP,VSL,VCL,ALH,BCF and STR,etc.The index was significantly reduced,but in the selenium intervention group,there was a clear upward trend.2The level of serum T in rats gradually decreased with the increase of exposure dose,and the difference between900 mg/kg DEHP exposure group and control group was statistically significant（P<0.05）.The level of serum T in each intervention group increased.The Se+900mg/kg intervention group was significantly different from the same dose of DEHP treated group（P<0.05）.Effects of sub-chronic exposure to DEHP and selenium on testis enzyme and oxidation markers in SD rats As the dose increased,SOD and GSH-Px in the testis of rats gradually decreased,and MDA gradually increased,compared with the control group.The difference was statistically significant（P<0.05,P<0.01）.The selenium intervention group had the opposite change.Compared with the same dose of DEHP exposure group,the difference was statistically significant（P<0.05,P<0.01）.The activity of SDH increased first and then decreased with the increase of exposure dose.The activity of LDH and ALP decreased gradually with the increase of the dose.The activity of ACP gradually increased with the increase of dose,and there was corresponding protection in the selenium intervention group.ability.ConclusionThrough sub-chronic exposure of rats to DEHP for 8 weeks and simultaneous intervention with yeast selenium,it was concluded that the liver,kidneys,spleen,testes,and epididymis of male rats are target organs for the toxic effects of DEHP.The epididymides are most sensitive to the toxic effects of DEHP,while selenium has a positive protective effect.The results of blood biochemistry and blood cell-related indicators in rats indicate that sub-chronic exposure to DEHP affects liver and kidney-related biochemical markers and white blood cells in rats（WBC,MONO,LUC）,red blood cells（RBC,HGB,HCT,MCH,HDW）and platelet PLT have universal toxic effects,selenium intervention has a certain protective effect;through detection of sperm motility and serum T levels in male rats.With the increase of DEHP exposure dose,the parameters of sperm motility in rats such as VAP,VSL,VCL,ALH,BCF and STR significantly decreased,but in the selenium intervention group,there was a clear upward trend.With the increase of the dose,the serum T level of rats gradually decreased,and the serum T levels in each intervention group increased,suggesting that DEHP disrupts sperm motility,disrupts hormone levels required for male reproduction,and selenium improves DEHP induction.The harmful effects have played a positive role;through the study of rat testicular function-related enzymes and lipid peroxidation injury indicators found that as the dose increased,the testis tissue of rats gradually reduced SOD,GSH-Px.MDA increased gradually,compared with the control group,the difference was statistically significant（P＜0.05,P＜0.01）.Selenium intervention group had the opposite change,and the difference was statistically significant compared with the same dose of DEHP exposure group.Significance（P＜0.05,P＜0.01）.The activity of SDH increased first and then decreased with the increase of exposure dose.The activity of LDH and ALP decreased gradually with the increase of the dose.The activity of ACP gradually increased with the increase of dose,and there was corresponding protection in the selenium intervention group.ability.It is suggested that DEHP or its metabolites may cause lipid peroxidation damage,which may be one of the important mechanisms that cause testicular tissue damage.Selenium,by exerting its antioxidant effects,protects the oxidative damage of testicular tissue caused by DEHP.In addition,DEHP may also affect the activity of these testicular function-related enzymes,resulting in insufficient supply of energy to the spermatogenic cells,resulting in reduced spermatogenic capacity,and selenium may prevent DEHP-induced infection to a certain degree by mentioning the activity of these enzymes.Testicular toxicity.